Isolation and Quantification of Microplastics from Pediatric Tonsils

Abstract

Background:
Microplastics have been identified in nearly every organ system, posing an increasing and
poorly characterized threat to human health. This project aims to isolate and quantify
environmentally acquired microplastics in pediatric tonsil samples. Tonsils provide a unique
opportunity for the isolation of microplastics. Not only are they highly vascularized, but they are
also exposed to microplastics through eating, drinking, and breathing.
Methods:
Pediatric tonsils, removed as standard of care, were obtained from a Stanford University
collaborator on an approved IRB protocol. Samples were digested in 10% KOH at 50C for 48hrs
with 30mins of ultrasonication at 24hrs (42kHz). Enzymatic digestion followed using collagenase
(1mg/mL), DNase (100μg/mL), and proteinase K (250μg/mL) for 24hrs. Post-digestion, samples
were stained with Nile Red (30μg/mL) to selectively visualize microplastics and filtered via
0.2μm aluminum oxide filters in an all-glass vacuum apparatus. Filters were imaged under
brightfield, Texas Red (ex-560nm), and CY3 (ex-540nm) fluorescence channels. Image
quantification was performed using R-statistical software to assess particle count, diameter, and
area.
Results:
The digestion protocol effectively degraded tissue while preserving suspected microplastics.
Filters captured fluorescently labeled particles larger than 0.2μm. Preliminary analyses revealed
fluorescently labeled particles in every sample (n=6). Across samples there was a mean particle
count of 268±274 and a median particle diameter ranging from 13.0μm to 15.6μm. No observed
correlation between tonsil mass (78.3±39.4mg) and particle number. In the tissue free negative
control, 39 particles were identified.
Conclusion/Impact:
Despite growing evidence of systemic microplastic contamination and an ever-increasing
exposure to plastics, reliable methods for isolating and characterizing environmentally acquired
microplastics remain limited. This work establishes an optimized protocol for non-destructive
isolation and quantification of microplastics from human tissue. The tonsil-isolated microplastics
will undergo further particle typing and be used in subsequent tissue culture models to better
understand microplastic exposure implications to human health.